Coacervation is the process by which an aqueous solution of a macromolecular colloid is separated into two liquid phases. One liquid phase, called the coacervate, is composed of many tiny colloid-rich droplets that are bound together. The other liquid phase, called the equilibrium liquid, is an aqueous solution of the coacervating agent.
When two or more oppositely-charged macromolecular colloids are used to form the coacervate, the process is termed complex coacervation. Colloids that bear a positive charge include gelatin and agar; colloids that bear a negative charge include carboxymethylcellulose and gum arabic. Depending upon each colloid's isoelectric point, dilution with water and/or adjustment of pH may be necessary for the particular colloids to be oppositely charged. These reactions must occur at a temperature above the gelling temperature for either colloid, otherwise the colloids will not be in a liquid phase and coacervation will not occur. When coacervation occurs in an environment that contains oil particles, the oil particles act as nucleating agents and the protein colloids deposit as a shell-like structure around each oil particle.
Encapsulating oil particles in the process of complex coacervation is well known in the prior art. U.S. Pat. No. 2,800,457 discloses oil-containing microscopic capsules and method of making them by complex coacervation. The '457 patent teaches dispersing a colloid in water, introducing an oil, forming an emulsion, dispersing a second colloid in water and mixing with the emulsion, and adjusting the pH and/or diluting with water to form a complex coacervate, all at a temperature above a gel point of the colloids, then cooling to cause a gel to form, followed by optional steps of hardening and cross-linking with formaldehyde or an equivalent. In one embodiment, gum arabic and gelatin are used to form a shell-like film of colloid material around an oil nucleus. Once the coacervate is formed, the mixture is allowed to stand for an hour at not over 25.degree. C., after which time the formation of capsules is complete. The capsules may then be used as desired or may undergo the optional hardening step. U.S. Pat. No. 2,800,458 similarly discloses a method for making oil-containing microcapsules. The '458 patent discloses the use of a salt solution to form the coacervate, while the '457 patent discloses either altering pH or diluting with water to form the coacervate.
Cross-linking of the protein shell of the complex coacervate renders the protein-encapsulated oil thermostable, since a protein containing cross-links is a stable structure. The use of known chemical cross-linking agents, such as formaldehyde or glutaraldehyde, to irreversibly cross-link the oil-containing capsules is disclosed in the prior art. Other cross linking agents such as tannic acid (tannin) or potassium aluminum sulfate (alum) are similarly known. The optional hardening step disclosed in both the '457 patent and the '458 patent consists of adjusting a suspension of capsular material to pH 9 to 11, cooling to 0.degree. C. to 5.degree. C., and adding formaldehyde.
Formaldehyde and glutaraldehyde, while effective chemical cross-linking agents, are toxic. Thus, oil capsules that have been cross-linked using such chemicals cannot be used for oils that may be applied to or ingested within a mammalian body without accounting for the toxicity. This severely limits the applications for such products.
Certain naturally-occurring enzymes are also good cross-linking agents. Such enzymes work by catalyzing the formation of bonds between certain amino acid side chains in proteins. In addition, because the enzymes are naturally occurring, encapsulated oils that are enzymatically cross-linked do not suffer from the problems inherent with formaldehyde and glutaraldehyde cross-linking, and hence may be ingested or applied without concern about the toxicity of the cross-linking agent. Because cross-linking is a enzyme catalyzed reaction, however, the proper environmental conditions must exist for optimum enzyme activity.
An enzyme that catalyzes protein cross-linking is transglutaminase (amine .gamma.-glutamyl transferase, EC 2.3.2.13). Transglutaminase catalyzes an acyl transfer reaction between .gamma.-carboxamide groups of glutamine residues in a peptide and various primary amines, frequently .epsilon.-amino groups of peptide-bound lysine residues. The result is a bond or cross-linkage between a glutamine residue in one protein molecule and a lysine residue in another protein molecule. For optimal activity, transglutaminase requires a divalent metal ion, usually calcium or magnesium, as a cofactor and a pH of around 7.
Japanese patent publication 5-292899 to Ajinomoto Inc. discloses the use of transglutaminase as a cross-linking agent in preparing microcapsules. The structure taught in that publication, however, is not believed to be a complex coacervate as defined by those skilled in the art. It is, rather, an enzyme-modified gelatin emulsion. Additionally, the 5-292899 publication discloses cross-linking at elevated temperatures. Molecular and/or particulate structures maintained at elevated temperatures are more fluid and less stable, resulting in cross-linking a molecule or particles of undefined structure. The Ajinomoto publication "Ajinomoto Co.'s Transglutaminase (TG)" discloses optimum cross-linking conditions for transglutaminase at pH 6-7 and elevated temperatures of 50.degree. C.